Bioassays using plasmonic scattering from noble metal nanostructures

ABSTRACT

The present invention relates to detecting and/or measuring scattering effects due to the aggregating metallic nanostructures or the interaction of plasmonic emissions from approaching metallic nanoparticles. The scattering effects may be measured at different angles, different wavelengths, changes in absorption and/or changes in polarization relative to changes in the distances between nanoparticles.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to assay methods and systems, and moreparticularly, to assay systems and methods using plasmonic emissionsgenerated by approaching or aggregating metallic surfaces.

2. Background of the Related Art

Dynamic Light Scattering (DLS), also referred to as photon correlationspectroscopy,¹ is the most widely used technique today for studyingcolloidal systems.²⁻⁵ It is a relatively fast technique, which canprovide absolute estimates of particle size and concentration for a widevariety of particles. However, the technique does have severallimitations.¹ These limitations include the low information content fromthe measured signal, the complexity of data analysis (this involves thenumerical inversion of a Laplace Transform¹) and the fact that both DLSand the other scattering techniques are not appropriate for very dilutesolutions of particles.²⁻⁵ Subsequently, this has been a limitation insensing biospecies at nanomolar and even lower concentrations.

Over the last several years, the use of both gold and silvernanoparticles in biological assays has dramatically increased. This hasbeen afforded by their very high molar absorption coefficients,^(6,7)which has enabled their use in many absorption-based (of light)nanoparticle assays.⁸⁻¹⁴ In addition to their high absorptioncross-sections, nanoparticles of gold and silver are also very efficientscatterers of light. Indeed a noble metal colloid's extinction spectrumis composed of both an absorption and scattering component, which iscontrary to how a typical fluorophores extinction spectrum isunderstood. Subsequently, light scattering by gold and silvernanoparticles can be detected at concentrations as low as 10⁻¹⁶ M⁶. Inaddition, it is well known that the light dependent scatteringproperties of a nanoparticle depend on their size, shape, compositionand the refractive index of the suspending medium.⁶

Typically, in cellular imaging today, fluorophores or even quantum dots,are used, which either contain some function groups to bind to expressedcellular surface features (receptors) or can even be transfected withinthe cells. This enables the cells to be readily imaged. However, oneparticular problem with using fluorophores is there inherent photoinstability, where most fluorophores typically photo degrade after about10³ excitation/emission event cycles. Thus, one constraint inimmunosensing is the detectability of the fluorophore.

Notably, light scattering by metallic structures in known but heretoforeseveral additional properties related to the light scattering frommultiple metallic structures have been ill explored for biosensingapplications. Thus, it would be advantageous to explore other scatteringproperties of nanoparticles and the interaction therebetween foraffinity biosensing, including the spatial distribution of light scatterand its subsequent polarization dependence; and the ability of for noblemetal nanostructures to dipole-couple over very large distances, therebyeffectively breaking the fluorescence resonance energy transfer (FRET)limit imposed by current organic fluorophores.

SUMMARY OF THE INVENTION

Surface plasmons are collective oscillations of free electrons atmetallic surfaces. These oscillations can give rise to the intensecolors of solutions of plasmon resonance nanoparticles and/or intensescattering. When a metallic nanoparticle is exposed to anelectromagnetic wave, the electrons in the metal (plasmons) oscillate atthe same frequency as the incident wave. Subsequently, the oscillatingelectrons radiate electromagnetic radiation with the same frequency asthe oscillating electrons. It is this re-radiation of light at the sameincident wavelength that is often referred to as plasmon scatter.⁶

Broadly the present invention relates to detecting and/or measuringscattering effects of plasmons from the surface of a metallic particleor from the interaction of plasmonic emissions from numerous approachingor aggregating metallic nanostructures. The scattering effects may bemeasured at different angles, different wavelength, changes inabsorption and/or changes in polarization relative to changes in thedistances between nanoparticles.

Notably, nanostructures are far superior to fluorophores in that theydon't photodegrade and have far greater emissions than that offluorophores. Further, nanostructures of gold and silver are veryefficient scatterers of light.

In one aspect, the present invention relates to the use of surfaceplasmons in a biosensing method for measuring concentration of ananalyte that induces colloidal aggregation, the method comprising:

-   -   (a) measuring the change in intensity of scattered light from        small colloids relative to the intensity of scattered light        measured for larger colloidal aggregates, wherein the intensity        of the scattered light is measured at two angles relative to the        incident light and a ratio is determined between the measured        values of the two angles, and wherein the ratio decreases as        aggregation increases.

In another aspect, the present invention relates to a biosensing methodfor measuring concentration of an analyte that induces colloidalaggregation, the method comprising:

-   -   (a) preparing metallic nanostructures comprising a noble metal        and at least partially coated with a binding component having an        affinity for the analyte, and wherein the colloids are at size        that scatters light according to the Rayleigh theory;    -   (b) exposing the metallic nanostructures with electromagnetic        radiation at a frequency that is absorbed and scattered;    -   (c) measuring the intensity of scattered light from the metallic        nanostructures, wherein the intensity of the scattered light is        measured at two angles relative to the incident light;    -   (d) contacting the metallic nanostructures with an analyte that        has an affinity for the binding component; measuring the        intensity of scattered light emitted from the metallic        nanostructures, wherein the intensity of the scattered light is        measured at the same two angles as in step (c); and    -   (e) determining a ratio between the measured intensity values at        the two angles, wherein the ratio approaches unity as        aggregation increases.

Preferably, the angles for measuring intensities include angle rangesfrom 30 to 175 degrees for one angle and from 185 to 350 degrees for thesecond angle, and more preferably from 40 to 160 degrees and 200 to 320degrees, and most preferably from 70 to 120 degrees and 230 to 300. Theelectromagnetic radiation is preferably applied by a monochromatic laserlight at a frequency similar to plasmon absorption maxima of thecolloids.

Preferably, the metallic nanostructures take the form of metallicparticles having any geometric shape such as triangular, elliptical,spheres; metallic islands or colloids; and/or porous matrix. In thealternative a continuous or semicontinuous metallic surface may be used.The metallic element may include any form of a noble metal such assilver, gold, platinum, copper or a combination thereof, and morepreferably the metallic material is gold or silver.

In a still further aspect, the present invention relates to using theemissions, due to the coupling of long range plasmonic scattering, frommetallic nanostructures to replace typically used fluorescence probes,thereby overcoming the shortcoming of fluorophores.

Another aspect of the present invention relates to a bioassay formeasuring concentration of receptor-ligand binding, the methodcomprising:

-   -   (a) preparing metallic nanostructures immobilized on a surface        wherein the metallic nanostructures have positioned thereon a        receptor molecule having affinity for the ligand;    -   (b) contacting the metallic nanostructures attached to the        receptor molecule with a sample suspected of comprising the        ligand of interest, wherein any ligand in the sample will bind        to the receptor molecule to form a receptor-ligand complex;    -   (c) contacting the receptor-ligand complex with a detector        molecule having affinity for the ligand to form a        receptor-ligand-detector complex, wherein the detector molecule        is attached to a metallic nanostructure, and wherein binding of        the ligand to the receptor forms a metal complex comprising        metallic nanostructures on opposing ends of        receptor-ligand-detector molecule complex;    -   (d) exposing the metal complex to electromagnetic radiation at a        frequency that is at least scattered by the metallic        nanostructures; and    -   (e) measuring an effect of coupling of plasmon scatter from the        metallic nanostructures of the metal complex, wherein the effect        comprises spectral shifts of wavelength absorption due to long        range plasmon coupling from the two opposing metallic        nanostructures, the ratio of scattered intensities from coupling        of the long-range plasmon scatter at two different wavelengths        and/or the reduction in polarization of the coupling of the long        range plasmon scatter when the metallic nanostructure move to        close proximity.

A still further aspect of the present invention relates to a sensingplatform for measuring the polarization of scattered light of plasmonicemissions from the metal complexes, wherein the polarization value isdecreased as the aggregation of the metal complexes increases. Further,the sensing platform may include measuring the change in intensity oflong range plasmon scattered light at two angles relative to theincident light and a ratio is determined between the measured values ofthe two angles, and wherein the ratio decreases as aggregation ofmetallic nanostructures increases.

Yet another aspect of the present invention relates to a biosensingmethod for measuring concentration of an analyte, the method comprising:

-   -   (a) preparing metallic nanostructures immobilized on a surface        wherein the metallic nanostructures are attached to a capture        molecule having affinity for the analyte, and wherein the        metallic nanostructures are sized to scatter light according to        the Rayleigh theory;    -   (b) exposing the metallic nanostructures and capture molecule        with electromagnetic radiation at a frequency that is absorbed        and/or scattered by the metallic nanostructures;    -   (c) measuring the intensity of scattered light, wherein the        intensity of the scattered light is measured at two angles        relative to the incident light;    -   (d) contacting the capture molecule with a sample suspected of        comprising the analyte of interest, wherein any analyte of        interest in the sample will bind to the capture molecule to form        a capture-analyte complex;    -   (e) contacting the capture-analyte complex with a detector        molecule having affinity for the analyte to form a        capture-analyte-detector complex, wherein the detector molecule        is attached to a metallic nanostructure to form a metal complex        comprising metallic nanostructures on opposing ends of        capture-analyte-detector molecule complex;    -   (f) measuring the intensity of scattered light from formed metal        complexes, wherein the intensity of the scattered light is        measured at the same two angles as in step (c); and    -   (g) determining a ratio between the measured intensity values at        the two angles, wherein the ratio approaches unity as        aggregation increases.

In as still further aspect the present invention relates to a biosensingmethod for measuring concentration of an analyte, the method comprising:

-   -   (a) preparing metallic nanostructures immobilized on a surface        or free in solution wherein the metallic nanostructures have        positioned thereon a capture/receptor molecule having affinity        for the ligand;    -   (b) exposing the metallic nanostructures and capture/receptor        molecule with electromagnetic radiation at a frequency that is        absorbed and scattered by the metallic nanostructures;    -   (c) measuring the absorption intensity and wavelength of        scattered light;    -   (d) contacting the metallic nanostructures attached to the        capture/receptor molecule with a sample suspected of comprising        the ligand of interest, wherein the ligand in the sample will        bind to the capture/receptor molecule to form a        capture/receptor-ligand complex;    -   (e) contacting the capture/receptor-ligand complex with a        detector molecule having affinity for the ligand to form a        capture/receptor-ligand-detector complex, wherein the detector        molecule is attached to a metallic nanostructure to form a metal        complex comprising metallic nanostructures on opposing ends of        the capture/receptor-ligand-detector molecule complex;    -   (f) measuring the absorption intensity and wavelength of        scattered light from formed metal complexes; and    -   (g) determining the spectral shift in absorption wavelength,        which is shifted as aggregation increases.

In a still further aspect, the present invention relates to an assayusing High Throughput Screening (HTS), the method comprising:

-   -   (a) providing a well plate used in HTS systems comprising a        multiplicity of wells;    -   (b) introducing metallic nanostructures into the wells, wherein        the metallic nanostructures are coupled to a binding receptor        having affinity for a target molecule    -   (c) introducing at solution suspected of including the target        molecule for binding to the binding receptor;    -   (d) introducing a detection molecule having affinity for the        target molecule, wherein the detection molecule comprises a        complement metallic nanostructure, wherein the complement        metallic nanostructure will be positioned traverse to the        metallic nanostructure on binding of the target molecule to the        detection molecule;    -   (e) applying electromagnetic energy; and    -   (f) measuring the plasmonic emissions from the system.

A further aspect of the present invention, relates to a kit fordetecting a target molecule in a sample, the kit comprising

-   -   (a) a container comprising a layer of immobilized metal        particles deposited on a substrate fabricated of a polymeric or        quartz material, wherein an immobilized probe is connected to        the metal particles and wherein the immobilized probe has an        affinity for the target molecule; and    -   (b) a detector molecule having an affinity for the target        molecule, wherein the detector molecule comprises a metallic        nanostructure and wherein the binding of the target molecule to        both the immobilized probe and detector molecule causes the two        metallic nanostructures to be positioned at a distance        sufficient for coupling of the plasmonic emissions from the        metallic nanostructure for subsequent measuring.

Other features and advantages of the invention will be apparent from thefollowing detailed description, drawings and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the coordinate system used to describe the geometricalarrangement of the illumination and detection systems (Top), and theapparatus used for measuring the angular dependence of colloidal scatter(Bottom).

FIG. 2 shows the normalized absorption spectra of different sized goldcolloids.

FIG. 3 shows the angular dependent scattering of 20 nm colloids using532 nm laser light as a function of colloid concentration (Top),enlarged 80-100 degree region (Top insert) and the I140/I90 intensityratio vs colloid concentration (Bottom).

FIG. 4 shows the angular dependent scattering from 0.8 nM differentsized gold colloids using 470 nm (Top), 532 nm (Middle) and 650 nm(Bottom) laser light.

FIG. 5 shows the intensity ratio at 140 and 90 degrees vs. colloid sizefor 3 different incident wavelengths.

FIG. 6 shows the scattered intensity at 140 degrees for different sizedcolloids as a function of time using 532 nm laser light.

FIG. 7 shows a model system (BSA-Biotin colloids crosslinked bystreptavidin) to demonstrate the utility of Angular-RatiometricPlasmon-Resonance based light scattering for affinity biosensing.

FIG. 8 shows the changes in absorption spectra of BSA-biotin colloidscrosslinked by streptavidin (Top) and the time dependent change inabsorption at 600 nm for a 15 nM streptavidin addition (Bottom).

FIG. 9 shows the angular dependent scattering from 20 nm BSA-biotincolloids crosslinked by streptavidin from 532 nm (Top) and 650 nm laserlight (Bottom).

FIG. 10 shows the scattered intensity ratio of I140/I90 from aggregatedBSA-biotin colloids for both 532 and 650 nm laser light as a function ofstreptavidin concentration.

FIG. 11 shows a simplified view of the intensities values for a goldparticle and the intensities due to colloidal aggregation showing thatas aggregation increases the ratio approaches unity.

FIG. 12 shows a sandwich immunoassay of the present invention.

FIG. 13 shows the shift in wavelength upon metal-metal/plasmon-plasmoncoupling.

FIG. 14 shows the ratio of plasmon scattering at different wavelengths.

FIG. 15 shows the decrease in polarization as the analyte increases dueto increase close proximity of the metallic structures.

FIG. 16 shows long range plasmon coupling for sensing platforms.

FIG. 17 shows the change in angular dependence of scatter upon abioaffinity induced aggregation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to affinity biosensing using plasmon lightscattering emissions from interacting metallic nanoparticles.

The term “biomolecule” means any molecule occurring in nature or aderivative of such a molecule. The biomolecule can be in active orinactive form. “Active form” means the biomolecule is in a form that canperform a biological function. “Inactive form” means the biomoleculemust be processed either naturally or synthetically before thebiomolecule can perform a biological function. Exemplary biomoleculesinclude nucleic acids, aromatic carbon ring structures, NADH, FAD, aminoacids, carbohydrates, steroids, flavins, proteins, DNA, RNA,oligonucleotides, peptide, nucleic acids, fatty acids, myoglobin, sugargroups such as glucose etc., vitamins, cofactors, purines, pyrimidines,formycin, lipids, phytochrome, phytofluor, peptides, lipids, antibodiesand phycobiliproptein.

The present invention provides enhanced emissions using metallizedislands of elliptical, spherical, triangular or rod-like forms. Inexemplary cases, the elliptical islands have aspect ratios of 3/2, andthe spherical colloids have diameters of 20-60 nm. However, theinvention is not limited to any particular geometry. Using known coatingtechniques, the placement of metallic islands could be controlledprecisely, as close as 50 nm apart.

Further, the metallic material may be in the form of a porous threedimensional matrix. The three dimensional matrix may be a nano-porousthree dimensional matrix. The metallic material may include metalcolloid particles and/or metal-silica composite particles. The metallicmaterial may comprise agglomerated metal particles and/or binary linkedparticles or metal particles in a polymer matrix. The three dimensionalmatrix may be formed from controlled pore glasses or using matricesassembled from the aggregation of silver-silica composites themselves.The matrices may be metallic nanoporous matrix, through which specieswill flow and be both detected and counted more efficiently.

Light sources used for applying electromagnetic energy can include anysource that may apply the necessary frequency or wavelength such as arclamps and lasers. Detectors can include photomultiplier tubes.Additionally, it is advantageous for the device to have a monochromatorso that specific wavelengths of light may be used to excite a moleculeor to detect emissions at a specific wavelength.

In one embodiment the metallic particle may be prepared by reduction ofmetal ions using various reducing agents, using technique known to oneskilled in the art. For example, sodium hydroxide may be added to arapidly stirred silver nitrate solution forming a brown precipitate.Then ammonium hydroxide is added to re-dissolve the precipitate. Thesolution is cooled and dried quartz slides are added to the beaker,followed by glucose. After stirring for 2 minutes, the mixture is warmedto 30° C. After 10-15 minutes, the mixture turns yellow-green andbecomes cloudy. A thin film of silver particles has formed on the slidesas can be seen from their brown green color. The slides are rinsed withpure water prior to use.

Colloids can be prepared as suspensions by citrate reduction metals.Preferred metals are silver and gold. Again, gold may be used because ofthe absorption of gold at shorter wavelengths. The size of the colloidsand their homogeneity can be determined by the extensive publications onthe optical properties of metal particles available and the effects ofinterface chemistry on the optical property of colloids.

Silver island films can be formed by a chemical reduction of a silversalt on the quartz surface, which are relatively simple to fabricate.

Metal particles can be bound to a surface by placing functional chemicalgroups such as cyanide (CN), amine (NH₂) or thiol (SH), on a glass orpolymer substrate. Metal colloids are known to spontaneously bind tosuch surfaces with high affinity. Positioning of the biomolecule ormetal particle at a desired distance can be achieved by using a film.The film may be a polymer film, a Langmuir-Blodgett film or an oxidefilm.

Metallic colloids (or various other non-spherical shapes/particles) mayalso be incorporated into organic polymers, covalently ornon-covalently, to form polymeric matrices, wherein the distance fromdiffusing species affords an increase in radiative decay rate and thus,an increase in quantum yield. Such polymeric matrices are ideal forsensing/flowing sensing applications of low concentration species.

In one embodiment, the application of low level microwave heating of thesample may be used to speed up any biological/biochemical kineticswithin the system. Notably, low level microwaves do not destroy ordenature proteins, DNA, or RNA, but instead heat the sample sufficientlyto provide for accelerated kinetics such as binding or hybridization. Inaddition, the microwaves are not scattered by the low density silvermetal, which is contrary to most metal objects, such as that recognizedby placing a spoon in a microwave oven.

Microwaves (about 0.3 to about 300 GHz) lie between the infrared andradiofrequency electromagnetic radiations. It is widely thought thatmicrowaves accelerate chemical and biochemical reactions by the heatingeffect, where the heating essentially follows the principle of microwavedielectric loss. Polar molecules absorb microwave radiation throughdipole rotations and hence are heated, where as non-polar molecules donot absorb due to lower dielectric constants are thus not heated. Thepolar molecules align themselves with the external applied field. In theconventional microwave oven cavity employed in this work, the radiationfrequency (2450 MHz) changes sign 2.45×10⁹ times per second. Heatingoccurs due to the tortional effect as the polar molecules rotate backand forth, continually realigning with the changing field, the molecularrotations being slower than the changing electric field. The dielectricconstant, the ability of a molecule to be polarized by an electricfield, indicates the capacity of the medium to be microwave heated.Thus, solvents such as water, methanol and dimethyl formamide are easilyheated, where as microwaves are effectively transparent to hexane,toluene and diethylether. For metals, the attenuation of microwaveradiation arises from the creation of currents resulting from chargecarriers being displaced by the electric field. These conductanceelectrons are extremely mobile and unlike water molecules can becompletely polarized in 10-18 s. In microwave cavity used in the presentinvention, the time required for the applied electric field to bereversed is far longer than this, in fact many orders of magnitude. Ifthe metal particles are large, or form continuous strips, then largepotential differences can result, which can produce dramatic dischargesif they are large enough to break down the electric resistance of themedium separating the large metal particles. Interestingly, and mostappropriate for the new assay platform described herein, small metalparticles do not generate sufficiently large potential differences forthis “arcing” phenomenon to occur. However, as discuss hereinbelow, thecharge carriers which are displaced by the electric field are subject toresistance in the medium in which they travel due to collisions with thelattice phonons. This leads to Ohmic heating of the metal nanoparticlesin addition to the heating of any surface polar molecules. Intuitively,this leads to localized heating around the silver nanostructures inaddition to the solvent, rapidly accelerating assay kinetics.

In the present invention, microwave radiation may be provided by anelectromagnetic source having a frequency in a range between 0.3 and 10GHz and a power level in a range between about 10 mwatts and 400 watts,more preferably from 30 mwatts to about 200 watts. Any source, known toone skilled in the art may be used, such as a laser that emits light,wherein light is used in its broad sense, meaning electromagneticradiation which propagates through space and includes not only visiblelight, but also infrared, ultraviolet and microwave radiation. Thus, asingle instrument placed above the surface of the assay can be used togenerate the microwave energy and energy to excite fluorescingmolecules. The light can be emitted from a fiber continuously orintermittently, as desired, to maintain the metallic particles at apredetermined temperature such that it is capable of increasing thespeed of chemical reactions within the assay system. In the alternative,microwave energy can be supplied through a hollow wave guide forconveying microwave energy from a suitable magnetron. The microwaveenergy is preferably adjusted to cause an increase of heat within themetallic material without causing damage to any biological materials inthe assay system.

One embodiment relates to a ratiometric approach to theangular-dependent light scattering from bioactivated and subsequentlyaggregated noble metal colloids. This new sensing system utilizes thechanges in particle scattering from very small colloids, which scatterlight according to traditional Rayleigh theory, as compared to thechanges in scattering observed by much larger colloidal aggregates,formed due to a bioaffinity reaction. These larger aggregates no longerscatter incident light in a Cos²θ dependence, as is the case forRayleigh scattering, but instead scatter light in a direction differentfrom the incident geometry, as shown in FIG. 17. By subsequently takingthe ratio of the scattered intensity at two angles, such as from 40 to160 degrees and 200 to 320 degrees, relative to the incident light, theassociation of metallic particles may be followed when crosslinkingagent is introduced.

As described above, horizontally polarized illumination of colloidsdemonstrates a Cos²θ dependence of plasmon scatter in the same plane forparticles that scatter in the Rayleigh limit. As shown in FIG. 17, 20 nmparticles, which initially scatter in the Rayleigh limit, change theirangular dependence of scatter upon a bioaffinity induced aggregation.Subsequently, the extent of forward scatter (180° with respect to theexcitation) significantly increases as the particles no longer purelyscatter in a Cos²θ dependence, but indeed scatter in the Mie limit. Bytaking the ratio of scattered intensities at two arbitrary wavelengths,one can readily follow a bioaffinity reaction. Interestingly, thedynamic sensing range is extended by both choosing to measure onescattered intensity at 90°, while also choosing particles that areinitially pure Rayleigh scatterers. Additionally, the measurements areindependent of total nanoparticle concentration, which is not the casefor other techniques such as dynamic light scattering. These findingssuggest a very simple approach for field deployable biosensors; usingeither LEDs or ambient room light, the observer simply takes the ratioof the scattered intensity at two angles to determine the concentrationof the biospecies of interest

This new model system can be potentially applied to many othernanoparticle assays and has many advantages over traditionalfluorescence sensing and indeed light-scattering approaches. Forexample, a single nanoparticle can have the equivalent scatteredintensity as 10⁵ fluorescing fluorescein molecules substantiallyincreasing detection; the angular distribution of scattered light fromnoble metal colloids is substantially easier to predict as compared tofluorescence; the scattered light is not quenched by biospecies; theratiometric measurements described here are not dependent on colloidconcentration as are other scattering techniques, and finally, the noblemetal colloids are not prone to photodestruction, as is the case withorganic fluorophores.

When a metallic nanoparticle is exposed to an electromagnetic wave, theelectrons in the metal (plasmons) oscillate at the same frequency as theincident wave.⁶ Subsequently, the oscillating electrons radiateelectromagnetic radiation with the same frequency as the oscillatingelectrons. It is this reradiation of light at the same incidentwavelength, which is often referred to as plasmon scatter.⁶ Thescattering of light by very small subwavelength sized particles, is welldescribed by Rayleigh theory.¹⁶ For incident light traveling along thex-axis, FIG. 1—top, and polarized in the y axis, the intensity of lightscattered, I_(scatt) in the direction θ by a homogeneous sphericalparticle with radius α, that is much smaller than the wavelength, λ, ofthe incident beam, is given by the Rayleigh expression,^(6,16)

$\begin{matrix}{I_{scatt} = {\frac{16\; \pi^{4}a^{6}n_{mod}^{4}I_{0}}{r^{2}\lambda^{4}}{\frac{m^{2} - 1}{m^{2} + 2}}^{2}{Cos}^{2}\vartheta}} & (1)\end{matrix}$

where I₀ is the incident intensity of monochromatic light, n_(med) isthe refractive index surrounding the particle, m is the refractive indexof the bulk particle material and r is the distance between theparticles and where the scattered light is detected. From FIG. 1—top thescattered light lies in the xy plane and is 100% polarized.Interestingly from FIG. 1 and Equation 1, the scattered light intensityis highest at the observation angles θ=0° and 180°, is zero at θ=90° and270°, and is proportional to Cos²θ at all other angles. This spatial orangular distribution of plasmon scatter is characteristic of an electricdipole emitter.⁶ Equation 1 is ideally suited for single particlescattering or for particle suspensions where the particles aresufficiently spaced so that interpretable perturbations and multiplescatterings are insignificant. For very dilute samples of multipleparticles, where the absorbance of the incident wavelength is less than0.005⁶ then the intensity of the scattered light can be obtained bymultiplying the single particle scattering intensity expressions by theparticle concentration, p, (particles/cm³). The intensity is thereforeproportional to the nanoparticle concentration and the dilute solutionhas same angular distribution of scatter, and degree of polarization, asan individual “Rayleigh particle.”⁶ However, for larger particles, wherethe size of the nanoparticle is ˜greater than 1/20^(th) the wavelengthof light, or for Rayleigh sized particles in close proximity to oneanother, the scattering properties no longer obey Rayleigh theory, butindeed can be described by Mie's theory.¹⁷ The angular dependence ofplasmonic scatter subsequently changes from the Cos²θ dependencedescribed by Equation 1 (Rayleigh limit), to a much more complex spatialdistribution of scatter, where the degree of forward scatter (i.e. 180°in our geometry, FIG. 1), increases with particle size (Mie limit).¹⁷

It is informative to briefly describe why the scattering from largerparticles is no longer described by Rayleigh theory. It was previouslymentioned that when a small particle is exposed to an electromagneticfield, whose wavelength is much larger than the diameter of theparticle, then the electrons in the nanoparticle all sense the samephase of the incident wave, and therefore all scatter light with thesame phase. In essence, the whole particle behaves as a largeoscillating dipole moment, a function of the collective electronoscillations (plasmons). However, for much larger particles then theelectrons on the particles can experience different phases, andtherefore can oscillate with different phases. This inherently leads tointerference of the light, which is scattered by the electrons fromdifferent parts of the particles. Subsequently, both the magnitude andangular distribution of the scattered light deviate from that expectedof a normal oscillating electric dipole. The Mie theory for lightscattering from large particles can be considered as light radiatingfrom oscillating electric dipoles, as well as magnetic dipoles,quadruples and other higher order magnetic multipoles.¹⁷ Scattered lightby Mie theory is well known and described by the following equation;^(∫)

$\begin{matrix}{I_{scatt} = {\frac{2\; \pi}{k^{2}}{\sum\limits_{n = 1}^{\infty}{\left( {{2\; n} + 1} \right)\left( {{a_{n}}^{2} + {b_{n}}^{2}} \right)}}}} & (2)\end{matrix}$

where k=2πn_(med)/λ. One can envision the different terms in the sum ascorresponding to different electric and magnetic multipoles and n is theterm index. The term with n=1 corresponds to the electric dipole. Thecoefficients an and bn are defined in terms of the Bessel and Ricattifunctions and in general are complex numbers depending on whether therefractive index of the particle is real or complex⁶. When the particleis much smaller than the wavelength of light, the most importantexpression in the Mie equation becomes that of the electric dipole, andthen the Mie equation reduces back to the Rayleigh expression.

To demonstrate the utility of this new sensing approach, the angulardependence of laser-light scatter from different sized gold colloids isdemonstrated herein. These colloids range in size from 20-200 nm, wherethe differences in their angular distribution of scatter serve toconfirm and support the bioaffinity solution based assay of the presentinvention, where small nanoparticles aggregate into much largerstructures using a crosslinking agent that facilitates such aggregation,such as shown in FIG. 7.

The angular distribution (spatial distribution) of scatter fromdifferent sized colloids is known to be markedly different,⁶ butheretofore has never been utilized for sensing. For small particleswhose diameters are less than 1/20^(th) the wavelength of light, thescatter of light is described well by Rayleigh theory,^(6,16) where asthe Mie theory¹⁷ describes the scattering from the much largerstructures. It is shown herein that the Cos²θ dependence of scatter fromsmall unaggregated particles in the Rayleigh limit, is no longer a validdescription of the scattering distribution for much larger, aggregatedparticles. In this regard, unaggregated colloids (monomers) were chosenwhose scatter can initially be well described by Rayleigh theory, butafter protein induced aggregation, now resides in the Mie limit forscattering. As mentioned, the initial choice of nanoparticle size isparamount for the biosensing platform described in this paper. Rayleightheory applies quite strictly to particles for which the radiusa<<<λ/(2πn _(med)|m|). For silver colloids and the wavelengths discussedhere, |m| is usually not greater than 4.⁶ For |m|=4, λ=532 and 650 nmand n_(med)=1.33, this expression yields particles with radii of 15.9and 19.4 nm respectively. According to Yguerabide,⁶ particles up toabout 40 nm are still considered to be in the Rayleigh limit.Subsequently for this work, 20 nm gold colloids were chosen, which canbe purchased monodispersed from Ted Pella, CA, USA.

In addition to “breaking” the Cos²θ angular dependence of Rayleighscatter by protein aggregation of the nanoparticles as an approach tosensing, the ratio of the scattered intensities at different angles wasdetermined, so that the measurements became independent of lightfluctuations, background light and most importantly nanoparticleconcentration, which is not the case with other light scatteringtechniques.¹⁻⁵ This subsequently allows the quantitative measurement ofthe concentration of protein in the model system, or potentially anyspecies that induces nanoparticle aggregation. Thus, thisangular-ratiometric plasmon-resonance based light scattering approachfor bioaffinity sensing, will serve as a model system which couldreadily be applied to the many other nanoparticle assays which have beendeveloped.⁸⁻¹⁴

To demonstrate the present sensing approach, whereby the angulardependence of plasmonic scatter changes upon colloidal aggregationinduced by a bioaffinity reaction, the scattering behavior of uncoatedgold colloid suspensions was initially studied. Notably, any noble metalmay be used to fabricate the nanostructures of the present invention, asdiscussed hereinabove.

FIG. 2 shows the normalized absorption spectra of different sized goldcolloids in citrate buffer. It can be clearly seen that the plasmonabsorption band at 520 nm for 20 nm colloids, shifting red, as well asbroadening as a function of size. Subsequently, for the angularscattering dependences discussed herein, monochromatic laser light at470, 532 and 650 nm was used because these frequencies are similar tothe plasmon absorption maxima of the colloids.^(14,18)

For bioaffinity sensing based on the angular dependence of plasmonscatter, it is important to understand the concentration dependence ofthe colloids on the scattering spatial distribution. FIG. 3 shows theangular scattering profiles for different concentrations of 20 nmcolloids using 532 nm monochromatic laser light. As predicted byequation 1, the Cos²θ dependence on scattering can clearly be seen inthese dilute samples. At angles approaching 0, 180 and 360 degrees, thescattering is the most pronounced, minimums in the scattering occurringat 90 and 270 degrees respectively. As expected the 2 sets of scatteringcurves in FIG. 3—top are mirror images of each other, and simply reflectthe 0-180 and 180-360 degree regions, as the fiber optic detector isrotated around the samples, c.f. FIG. 1—bottom. Approaching 0, 180 and360 degrees, the detector is rapidly saturated and hence the intensityvalues at 0, 180 and 360 degrees were not measurable. In fact, thedynamic range of scattering presented in FIG. 3—top is a compromisebetween the laser power used, and being able to detect both the minimumand maximum scattering intensities. Preferably, measurements aredetermined for scattering intensities at angles from 40 to 160 and 200to 320 degrees.

Rayleigh theory for the scattering of light by dilute solutions of smallcolloids predicts that the scattering intensity at a given angle,increases as a function of colloid concentration. This can be clearlyseen in FIG. 3—top insert, which is simply the enlarged 80-100 degreeregion. However, by taking the ratio of any two intensities, in thispresent situation 90 and 140 degrees, then the scattered intensitiesbecome independent of the colloid concentration. In addition, theseangular ratiometric measurements are independent of excitation (lightfluctuations) or detector drifts as well as background room light,notable features for biosensing.

FIG. 3—bottom indeed demonstrates this sensing strategy and shows anangular-ratiometric plot of intensities recorded at 140 and 90 degreesas a function of 20 nm gold colloid concentration. The linearity of theplot shows that the concentration of the colloids does not change thespatial distribution of the scatter, which is a most importantconsideration for sensing applications. In addition, the concentrationrange studied, typically reflects that used in colloidal plasmonabsorption type biosensing assays¹⁹⁻²¹.

FIG. 4 shows the angular dependent scattering from 0.8 nM differentsized gold colloids using 470, 532 and 650 nm laser light, top to bottomrespectively. There are two main features to these plots. Firstly, thecolloidal size dependence of scattering at a given incident wavelength,and secondly, the differences in scattering distributions for a givencolloid size using different incident wavelengths. At a given incidentwavelength, all the plots show a size dependence on scatteringdistribution, FIG. 4. These results show that the scattering from 20 nmcolloids typically follows a Cos²θ dependence as depicted by Rayleightheory, i.e. equation 1. As the colloidal size increases, the angularscattering profiles become much more complex and are no longer describedby the Rayleigh expression but can indeed be described by Mie'stheory¹⁷. In Mie's theory, the degree of forward scatter increases asthe particle size increases.¹⁷ From FIG. 4—middle, it can be clearlyseen that the width of the scattering spectrum at 180 degrees increaseas a function of colloidal size. In the presently described system, 180degrees is the angle of forward scatter, as shown in FIG. 1 and FIG. 17.

Subsequently, by taking the ratio of the scattered intensities atdifferent angles, such as 90 and 140 degrees as a function of goldcolloid size, FIG. 5, it can be seen how the size distribution ofscatter is changing. For small colloids, I₁₄₀/I₉₀ ratio is quite large,in the range 3-7, as the scattering follows a Cos²θ dependence. However,for much larger colloids the discrete Cos²θ scattering distributions arelost, the scattering no longer follows a Cos²θ dependence and the ratiobecomes close to unity, as shown in FIG. 5.

For the presently described bioaffinity sensing scheme, the initial goldcolloid size was chosen so that it would be in the Rayleigh limit, i.e.within the range of 10 nm to 40 nm, and more preferably from about 20 nmto 30 nm. Upon affinity induced aggregation of the colloids, thescattering distributions become increasingly more complex as a functionof protein addition, the scattering no longer following a Cos 2θdependence. Similar to the unmodified colloids, the I₁₄₀/I₉₀ ratiochanges, and can subsequently be correlated with protein concentration,or indeed any analyte or biospecies which can induced colloidalaggregation. Importantly, the dynamic range of the sensing strategymanifests itself in being able to aggregate particles that initiallyscatter in the Rayleigh limit, into the Mie limit after aggregation,c.f. the range shown in FIG. 5.

From FIGS. 4 and 5 it can be seen that the scattering distribution andtherefore the I₁₄₀/I₉₀ ratio for a given colloid size changes withincident wavelength respectively. Unlike the colloidal size dependencefor a fixed incident wavelength, these trends are much more complex tointerpret and are due to both the absorption and scattering componentsof a colloid's extinction spectrum,⁶ in addition to the position of theplasmon absorption maximum.^(6,14,18) For the sensing scheme describedherein, both 532 and 650 nm incident wavelengths were chosen. This isbecause 20 nm colloids have a plasmon absorption maxima at ˜520 nm,¹⁴⁻¹⁸and the potential applications of this technology to biological sensingand the respective need to alleviate biological auto-fluorescence byusing longer incident wavelengths.²²

Finally, to investigate both the photostability of the colloids, as wellas to ascertain whether the colloids would settle out of solution as afunction of time, the scattering intensity was monitored as a functiontime using 532 nm incident light measured at 140 degrees and shown inFIG. 6. For all colloid sizes it was found that the intensity remainedconstant over the 30 minute measurement period. This was particularlyencouraging and demonstrates that both the colloids do not settle fromsolution during measurements, and that the laser powers employed(several mW) do not alter the shape of the colloids, as has beenreported by some authors, but for higher incident laser powers.²³ FromFIG. 6, it can be seen that the colloids are photostable, more so thantraditional fluorophores, which are prone to photo degradation,²² theirscattering distributions not changing as a function of time.

To demonstrate the utility of the described system, a model proteinsystem was chosen as shown in FIG. 7. Biotinylated bovine-serumalbumin-coated 20 nm colloids (BSA-colloids) can be readily prepared,which cluster by the addition of the tetravalent protein streptavidin,¹⁹and the association of biotin and streptavidin is very strong,¹⁹eliminating the possibility of back disassociation reactions tocomplicate our model system's kinetics.

FIG. 8—top shows the normalized absorption spectra of BSA-colloids as afunction of streptavidin addition. The absorption spectra were takenafter completion of the aggregation, which was typically 45 minutes foreach sample. FIG. 8—bottom shows the time dependent change in absorptionat 600 nm for a 15 nm streptavidin addition. After 2000 seconds, it canbe seen that the reaction is essentially >90% complete.

Identical concentration and compositions of BSA-colloids weresubsequently incubated with different concentrations of streptavidin for45 minutes, the angular scattering profiles then taken using both 532(FIG. 9—top) and 650 nm (FIG. 9—bottom) incident light, in an analogousmanner to the virgin colloids described earlier. From FIG. 9 it can beclearly see that the streptavidin induced association reaction changesthe angular scattering profile from a relatively simple Cos²θ dependencein the absence of streptavidin, to one much more complex, with a higherdegree of forward scatter. These changes are consistent with a change inscattering from Rayleigh like particles to particles in the Mie limit asdepicted by equations 1 and 2. Importantly, by taking the I₁₄₀/I₉₀intensity ratio, the BSA-colloid association reaction was followed as afunction of streptavidin addition as shown in FIG. 10. Using this modelsystem, a notable change was observed in the I₁₄₀/I₉₀ intensity ratiousing 532 and 650 nm incident light. While a relatively simple modelsystem was chosen to demonstrate this sensing approach, this approachcan be applied to monitor many other nanoparticle biologicalassociation/disassociation reactions.

Thus, one embodiment of the present sensing system includes a newapproach to bioaffinity sensing wherein nanoparticles are used insteadof fluorophores for sensing, and this approach provides advantages overother scattering-based techniques including:

-   -   simplification of instrumentation by using a laser and measuring        the ratio of the scattered light intensity at two unique angles;    -   the ratio of the scattered intensity at two angles which is        independent of nanoparticle concentration;    -   use of the metallic nanoparticle are not prone to        photodestruction with the use of the intense illumination        intensities in contrast to fluorophores; and    -   the lifetime of plasmon scatter is usually less than 1 ps, where        as fluorescence lifetimes are usually on a nanosecond timescale,        as such, plasmon light scatter is not prone to quenching by        biospecies, unlike many fluorophores.^(6,22)

In another embodiment, a receptor molecule may be fused to a noble metalnanostructure that can be free in solution or immobilized on a glass orpolymeric smooth surface, wherein a ligand in a sample can bind to thereceptor molecule. Importantly, instead of using a fluorescing detectorbiomolecule to bind to the ligand to determine presence of such a ligandin the assay, the present invention provides for a detector moleculethat is attached to a metallic nanostructure and has affinity for theligand at a position different from that of the receptor molecule. Whenthe system is irradiated with electromagnetic energy, both the noblemetal nanostructure and the metal on the detector molecule radiate longrange plasmonic emissions that are coupled, as shown in FIG. 16 and canbe subsequently measured using several different sensing platformsincluding measuring changes in plasmon absorption, intensity of scatter,angular dependence of scatter or polarization based scatter.

For example, when the two metal nanostructures approach, thepolarization of plasmonic scatter changes due to metal aggregationinduced by a bioaffinity reaction. The difference of the polarization ofthe coupled plasmonic scatter is compared to a control fornon-aggregated nanostructures. As shown in FIG. 15, as the binding ofligand/analyte to the capture/receptor increases, the polarizationdecreases. Thus, this decrease in polarization can be used to determinethe level of concentration of ligand/analyte when compared to a controlsystem. As formation of the receptor-ligand-detector increases withaggregation of metallic nanostructures, the polarization decreasesproportional to the concentration of binding ligand.

Upon aggregation of the nanoparticles, the solution polarization rapidlydecreases due to near-field plasmon coupling. Interestingly, by choosingparticles that initially scatter incident light in a Rayleigh manner,the present inventor has been able to show that the spatial distributionof polarized scatter also changes upon particle aggregation as theparticles now scatter in an increased forward direction (i.e. in the Mielimit). With an initial solution optical density of ≈1, significantdepolarization occurs at angles greater than 140°, less than 220° andmaximum around 180° from the incident excitation. Subsequently, thisapproach allows the determination of solution protein or analyteconcentrations using polarized scatter, the dynamic sensing rangedetermined by the angle of observation.

For the presently described sandwich assay sensing scheme, the initialnoble metal immobilized nanostructures were chosen so that it would bein the Rayleigh limit, i.e. 20 nm. Upon receptor/ligand inducedaggregation of the two metal structures at opposing ends of thereceptor/ligand/detector complex, the scattering distributions becomeincreasingly more complex as a function of ligand binding, and thescattering no longer following a Cos 2θ dependence. The polarizationchanges, and can subsequently be correlated with ligand concentration.Any analyte or biospecies which can induce the approach of the metallicstructures to cause interaction between the structures and aggregationmay be used in the present invention. Importantly, the dynamic range ofthe sensing strategy manifests itself in being able to aggregateparticles that initially scatter in the Rayleigh limit, into the Mielimit after aggregation.

The angle-dependent long-range polarized-scattering from metallicsurfaces can be measured using g using an X-Y rotating stage (EdmundOptics), that was modified to hold a cylindrical cuvette (a thin walledNMR tube), with a fiber optic mount, as shown in FIG. 1. The metallicstructures can be illuminated with vertically polarized laser sourceswith a neutral density filter being used to adjust the laser intensity.The angle-dependent vertically polarized scattered light from themetallic surfaces can be collected through a dichroic sheet polarizer(Edmund optics) into a 600 micron broad wavelength fiber that wasconnected to an Ocean Optics HD2000 spectrofluorometer. Thephotostability aggregation of metallic surfaces can be measured bysimply observing the polarized scattered intensity at different angles,such as 90 or 140 degrees for a specific length of time, such as 30 or45 minutes.

Notably, the present invention provides for the application of plasmonscatter and the measurement of distances in the range 10-300 nm forbiological systems. Today, optical distance measurements less than 10 nmare undertaken using FRET between a fluorescent donor and an acceptor.Distances ranging from macroscopic to about λ/2, typically about 300 nm,can be measured using confocal, multiphoton and/or laser scanningmethods but these systems are not readily compatible with biologicalspecies, such as live cells. Thus, long-range FRET utilizing plasmonicsmay be possible, based on the changes in the scattering, absorption andpolarization properties of suitably sized colloids. Interestingly, thecoupling distance (transfer distance in FRET) is dependent on thewavelength of light and the initial choice of colloid size. Thisapproach may be of significant importance for studying macromoleculardynamics and particularly in immunoassays, which typically havedimensions far too large for classical FRET.

Another sensing platform that is applicable to the coupling oflong-range plasmonic scatter from two separate and distinct metallicsurfaces that approach and interact includes wavelength ratiometricplasmon scattering. Specifically, measuring the intensity of the coupledlong range scattered light from the opposing ends of the metal complexcan be measured at two separated angles and determining the ratio ofsame. Importantly, as more receptor-ligand complexes form and formationof metal complexes increases the ratio of the scattered light at the twoseparate angles approaches unity as discussed previously relating toFIG. 5.

This approach is unique in that the measurements are independent of bothexcitation light fluctuations, as well as the concentration of thecolloidal sensing species. As shown in FIG. 14 by exciting the surfacewith two different laser lines at different wavelengths, or even whitelight illumination, and then taking the ratio of the scatteredintensities (at 2 unique wavelengths, λ₁ and λ₂) the concentration ofthe ligand/analyte can be ratiometrically determined.

FIG. 13 shows yet another sensing platform to determine the level ofligand/analyte binding. In the sandwich immunoassay shown in FIG. 12,the presence of an analyte facilitates bringing a noble-metalnanostructure in close proximity to a metallic surface. Long-rangeplasmon coupling occurs (up to 1000 nm), far-greater than anyfluorescence based coupling (up to 10 nm max for resonance energytransfer based assays), causing a change in surface color due to thechange in absorption at different wavelengths, depending on theconcentration of the analyte of interest. When the analyte of interestbinds to the surface antibodies as shown in FIG. 12, followed by thesecond antibody which is labeled with a metallic nanostructure, then aspectral shift in the absorption occurs as shown in FIG. 13.

Example Materials and Methods Materials

Colloidal gold dispersions (20, 40, 100 and 200 nm) were purchased fromTed Pella. Glycerol, sodium phosphate monobasic, phosphate bufferedsaline (PBS), streptavidin, biotinamidocaproyl labeled bovine serumalbumin (biotinylated BSA) and standard glass NMR tubes (5 mm, series300) were purchased from Sigma-Aldrich. All chemicals were used asreceived.

Methods

Preparation of Biotinylated BSA-Coated 20 nm Gold Colloids

The surface modification of 20 nm gold colloids was performed using anadapted version of the procedure found in the literature.¹⁹ In thisregard, 5 mL of the gold colloid solution was mixed with 0.05 mL aqueoussolution of biotinylated BSA (1.44 mg/mI), and this mixture wasincubated at room temperature for 2 hours. The gold colloid/biotinylatedBSA mixture was then centrifuged in an Eppendorf centrifuge tubeequipped with a 100,000 MW cut-off filter for 10 minutes, using anEppendorf microcentrifuge at 8,000 g, to separate the biotinylatedBSA-coated gold colloids from the excess biotinylated BSA. Thesupernatant was carefully removed, and the pellet containing thebiotinylated gold colloids was resuspended in 10 mM sodium phosphatebuffer (pH 7). This was subsequently used in the aggregation assays.

Aggregation Assay Using Biotinylated Gold Colloids and Streptavidin

The model aggregation assay, used to demonstrate the utility of thepresent invention, was performed by mixing biotinylated gold colloids(20 nm) with increasing concentrations of streptavidin in a quartzcuvette. In this regard, a 1000 nM stock solution of streptavidin(prepared in PBS based on the specifications provided by manufacturer,E1% at 282 nm=31.0) was added to 0.5 mL of biotinylated gold colloidsamples and incubated at room temperature for 30 minutes. In order toachieve the desired final streptavidin concentrations, predeterminedvolumes of streptavidin stock solution were used. The degree ofaggregation was measured by recording the absorption spectrum of eachsample (as with all other absorption measurements), using a Varian Cary50 spectrophotometer.

Aggregation Assay

The angle-dependent scattering from gold colloids of various sizes andthose used in the aggregation assay were measured using an X-Y rotatingstage (Edmund Optics), that was modified to hold a cylindrical cuvette(a thin walled NMR tube), with a fiber optic mount (FIG. 1—bottom). Thegold colloids were illuminated with three different vertically polarizedlaser sources: 470, 532 and 650 nm, a neutral density filter being usedto adjust the laser intensity. The angle-dependent vertically polarizedscattered light from the gold colloids was collected through a dichroicsheet polarizer (Edmund optics) into a 600 micron broad wavelength fiberthat was connected to an Ocean Optics HD2000 spectrofluorometer. Thephotostability of 20, 40 and 200 nm gold colloids, under constantillumination with a 532 nm laser, was measured by simply observing thescattered intensity at 140° for 30 minutes.

REFERENCES

The contents of all cited references are hereby incorporated byreference herein for all purposes.

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1. A biosensing method for measuring concentration of an analyte thatinduces colloidal aggregation, the method comprising: measuring thechange in intensity of scattered light from small colloids relative tothe intensity of scattered light measured for larger colloidalaggregates, wherein the intensity of the scattered light is measured attwo angles relative to the incident light and a ratio is determinedbetween the measured values of the two angles, and wherein the ratiodecreases as aggregation increases.
 2. The method according to claim 1,wherein the analyte is an amino acid sequence or nucleotide sequence. 3.The method according to claim 1, wherein the small colloids are in arange from about 10 nm to 40 nm.
 4. The method according to claim 1,wherein the small colloids are of a size that obeys Rayleigh theory ofscattered light.
 5. The method according to claim 1, wherein the smallcolloids are gold or silver particles.
 6. The method according to claim1, wherein the small colloids are coated with a binding component havingan affinity for binding with the analyte.
 7. The method according toclaim 1, wherein the angles for measuring intensities comprise from 40to 160 degrees for one angle and from 200 to 320 degrees for the secondangle.
 8. A biosensing method for measuring concentration of an analytethat induces colloidal aggregation, the method comprising: (a) preparingcolloids comprising a noble metal and at least partially coated with abinding component having an affinity for the analyte, and wherein thecolloids are sized to scatter light according to the Rayleigh theory;(b) exposing the colloids with electromagnetic radiation at a frequencythat is absorbed and scattered; (c) measuring the intensity of scatteredlight from colloids, wherein the intensity of the scattered light ismeasured at two angles relative to the incident light; (d) contactingthe colloids with an analyte that has an affinity for the bindingcomponent; measuring the intensity of scattered light from the colloids,wherein the intensity of the scattered light is measured at the same twoangles as in step (c); and (e) determining a ratio between the measuredintensity values at the two angles, wherein the ratio approaches unityas aggregation increases.
 9. The method according to claim 8, whereinthe electromagnetic radiation is applied by a monochromatic laser lightat a frequency similar to plasmon absorption maxima of the colloids. 10.A bioassay method for measuring concentration of receptor-ligandbinding, the method comprising: (a) preparing metallic nanostructuresimmobilized on a surface wherein the metallic nanostructures havepositioned thereon a receptor molecule having affinity for the ligand;(b) contacting the metallic nanostructures attached to the receptormolecule with a sample suspected of comprising the ligand of interest,wherein any ligand in the sample will bind to the receptor molecule toform a receptor-ligand complex; (c) contacting the receptor-ligandcomplex with a detector molecule having affinity for the ligand to forma receptor-ligand-detector complex, wherein the detector molecule isattached to a metallic nanostructure, wherein binding of the ligand tothe receptor forms a metal complex comprising metallic nanostructures onopposing ends of receptor-ligand-detector molecule complex; (d) exposingthe metal complex to electromagnetic radiation at a frequency that isabsorbed and/or scattered by the metallic nanostructures; and (e)measuring an effect of coupling of long range plasmon scatter from themetallic nanostructures of the opposing ends of the metal complex,wherein the effect includes spectral shifts of wavelength absorption dueto long range plasmon coupling from the two opposing metallicnanostructures, measuring the ratio of scattered intensities fromcoupling of the long-range plasmon scatter at two unique wavelength ormeasuring the reduction in polarization of the coupling of the longrange plasmon scatter when the metallic nanostructure move to closeproximity.
 11. The bioassay method according to claim 10, comprisingmeasuring the polarization of scattered light of plasmonic emissionsfrom the metal complexes, wherein the polarization value is decreased asthe aggregation of the metal complexes increases and the intensity ofthe radiation increases as aggregation increases.
 12. The bioassaymethod according to claim 10, wherein the surface supporting themetallic nanostructures is glass, quartz, polymeric or a combinationthereof.
 13. The bioassay method according to claim 10, wherein themetallic nanostructures are fabricated from gold or silver.
 14. Abiosensing method for measuring concentration of an analyte, the methodcomprising: (a) preparing metallic nanostructures immobilized on asurface wherein the metallic nanostructures are attached to a capturemolecule having affinity for the analyte, and wherein the metallicnanostructures are sized to scatter light according to the Rayleightheory; (b) exposing the metallic nanostructures and capture moleculewith electromagnetic radiation at a frequency that is absorbed andscattered by the metallic nanostructures; (c) measuring the intensity ofscattered light, wherein the intensity of the scattered light ismeasured at two angles relative to the incident light; (d) contactingthe capture molecule with a sample suspected of comprising the analyteof interest, wherein any analyte in the sample will bind to the capturemolecule to form a capture-analyte complex; (e) contacting thecapture-analyte complex with a detector molecule having affinity for theanalyte to form a capture-analyte-detector complex, wherein the detectormolecule is attached to a metallic nanostructure to form a metal complexcomprising metallic nanostructures on opposing ends ofcapture-analyte-detector molecule complex; (f) measuring the intensityof scattered light from formed metal complexes, wherein the intensity ofthe scattered light is measured at the same two angles as in step (c);and (g) determining a ratio between the measured intensity values at thetwo angles, wherein the ratio approaches unity as aggregation increases.15. The bioassay method according to claim 14, wherein the angles formeasuring intensities comprise from 40 to 160 degrees for one angle andfrom 200 to 320 degrees for the second angle.
 16. The bioassay methodaccording to claim 14, wherein the electromagnetic radiation is appliedby a monochromatic laser light at a frequency similar to plasmonabsorption maxima of the metal complexes.
 17. A biosensing method formeasuring concentration of an analyte, the method comprising: (a)preparing metallic nanostructures immobilized on a surface wherein themetallic nanostructures have positioned thereon a receptor moleculehaving affinity for the ligand; (b) exposing the metallic nanostructuresand receptor molecule with electromagnetic radiation at a frequency thatis absorbed and scattered by the metallic nanostructures; (c) measuringthe absorption intensity and wavelength of scattered light; (d)contacting the metallic nanostructures attached to the receptor moleculewith a sample suspected of comprising the ligand of interest, whereinany ligand in the sample will bind to the receptor molecule to form areceptor-ligand complex; (e) contacting the receptor-ligand complex witha detector molecule having affinity for the ligand to form areceptor-ligand-detector complex, wherein the detector molecule isattached to a metallic nanostructure to form a metal complex comprisingmetallic nanostructures on opposing ends of receptor-ligand-detectormolecule complex; (f) measuring the absorption intensity and wavelengthof scattered light from formed metal complexes; and (g) determining thespectral shift in absorption wavelength, which is shifted as aggregationincreases.
 18. The bioassay method according to claim 17, wherein theabsorption of the coupling of plasmonic scattering causes a change insurface color.
 19. The bioassay method according to claim 17, whereinthe spectral shift correlates to the concentration of formed metalcomplexes.
 20. The bioassay method according to claim 17, wherein themetallic nanostructures are from about 10 nm up to 1000 nm.
 21. Thebioassay according to claim 17, wherein the metallic nanostructures arefabricated from a noble metal.